It is axiomatic that the quality of water is essential for human health. The increasing worldwide contamination of freshwater systems with thousands of industrial and natural chemical compounds is one of the key environmental problems facing humanity today, where pathogens in water cause more than 2 million deaths annually. With more than one-third of the accessible and renewable freshwater used for industrial, agricultural, and domestic applications, pollution from these activities leaves water sources contaminated with numerous synthetic and geogenic compounds. In addition, natural disasters can result in large-scale disruptions of infrastructure, resulting in compromised water quality. Diarrheal disease caused from such disasters may be a major contributor to overall morbidity and mortality rates. Thus, the cleanliness and safety of public water sources has prompted researchers to look for rapid and sensitive indicators of water quality. Whereas most water filtering systems are quite efficient in removing large-size contaminants, smaller particles frequently pass through. These contaminants are often poorly soluble in water and present in quantities of less than 1 nM.
Modern analytical tools have become extremely efficient in the detection and analysis of chemical compounds. For example, liquid chromatography coupled with detection by tandem mass spectrometry has been used for detection of trace pharmaceuticals and other wastewater-derived micropollutants. Although such methods are very powerful in identifying trace pollutants, cost prohibits their widespread use by environmental researchers and, most importantly, prevents real-time analysis of water quality. Other techniques using bench top gas chromatography-mass spectrometry have also been demonstrated as viable methods for detection of basic pharmaceuticals with reduced cost. Despite this, these methods are still cost prohibitive, can hardly be used in field studies, and are unlikely to ever be used for real-time quality control.
In addition to pharmaceutical and other synthetic pollutants such as pesticides, animal and human waste (e.g., feces and urine) are an enormous source of water contamination that can be found in both recreational and source waters. These discarded wastes, when released into water, can carry a variety of diseases such as polio, typhoid, and cholera. In extreme cases pollution of an ecosystem can result in environmental crises, such as, for example, devastation to the aquatic population, red-tide blooms, as well as beach closings. Molecular methods based on polymerase chain reactions are commonly used to monitor viral, bacterial, and protozoan pathogens in wastewater. Microbiological indicators such as fecal coliforms, Escherichia coli, and Etherococci are indicators most commonly used to analyze and evaluate the level of fecal contamination. However, the suitability of these indicators has been questioned, and it takes a substantial amount of time from the extraction of a water sample for analysis to the moment when results are ready.
An alternative indicator that has been shown to be helpful in detection of waste in water supplies is urobilin. Urobilin is one of the final byproducts of hemoglobin metabolism, and is excreted in both the urine and feces of many mammals, including humans and common livestock (e.g., cows, horses, and pigs). In addition, as urobilin can be indicative of disease such as hepatic dysfunction or jaundice, an ultrasensitive technique for detection and quantification of this biomarker in solution has both diagnostic and environmental applications.
Urobilin detection in solution has previously been demonstrated using the formation of a phosphor group from the combination of urobilins and zinc ions. Normal heme catabolism results in the production of bilirubin, a red product, which is then broken down into two end products, stercobilin, the bile pigment found in fecal material, and urobilin, the yellow pigment found in urine. Both urobilin and stercobilin have been shown to be viable biomarkers for detection of fecal pollution levels in rivers.
Fluorescent detection of urobilin in urine has been demonstrated based on Schlesinger's reaction in which an urobilinogen-zinc chelation complex exhibits a characteristic green fluorescence when excited by blue light. Methods for detection of urobilinoids using high-performance liquid chromatography with a reversed-phase column and an ultraviolet detector have also been presented; however, the initial sensitivity of this method proved insufficient for clinical analysis. An increase in detection sensitivity of this methodology has been reported, but only to detection levels of 1.5 nM, where efficient excitation and collection of the fluorescent signal remained the limiting factor.
Traditional epiillumination fluorescence spectroscopy systems use an objective lens to focus excitation light into the sample and collect the fluorescence emission. In such a configuration, the signal generated is limited to the focal volume of the optics. In addition, the generated signal is diffusive in nature; only a small fraction of the total emitted light is collected. Because only a small volume of a sample can be probed at any given time with such a configuration, detection of subnanomolar concentrations remains difficult as these measurements are akin to single molecule detection. Thus, a method that could allow for probing a larger volume of a sample while also providing means for collecting more of the fluorescence emission could greatly enhance the ability to detect subnanomolar concentrations of urobilin.